Targeted Subcellular Delivery of Oligonucleotides and Proteins. Nucleic acid-based therapeuticals and other macromolecules, as a result of astounding advances in the fields of molecular/cellular biology and genomics, are striving to evolve from conceptually-satisfying experimental approaches to a clinical reality. However, successful implementation of these goals relies on overcoming the accompanying greater difficulties in delivering these large molecular compounds to their cellular and subcellular target sites in therapeutically effective modes and quantities. The major goal of this proposal is to characterize and develop efficient delivery strategies and delivery vectors for the realization of therapeutic goals of oligonucleotide (ON)- and protein-based drugs. One of the powerful applications of ONs is their usage as ligands for a variety of receptors, especially those of the immune cells. Considering the enormous importance of prophylactic and therapeutic usage of vaccines against viral infections and tumors, the powerful capacity of ONs in enabling and tailoring the immunologic outcome of vaccination will be explored;this proposal is focused on an efficient delivery system that can put ONs and protein antigens into the appropriate subcellular compartments of desired cell types. To test the delivery capabilities of the proposed delivery systems, we will first examine the hypothesis that endosome-permeabilizing listeriolysin O (LLO) co-encapsulated with immunostimulating sequence ONs (ISS-ONs) inside antigen-carrying delivery systems can deliver a significant extent of antigen to the cytosol (i.e., MHCI pathway of antigen presentation) as well as MHCII compartments while enhancing the delivery of ISS-ONs to the cognate receptors in the endocytic vesicles. The antigen- and ISS-ON-carrying LLO-liposomes will be used to test the immuno-stimulating and -modulating effects in comparison with control immunizations. The role of LLO, in addition to its ability to enhance cytosolic delivery via endosomolysis, in the modification of endosome maturation and slowing down its progression into lysosomes will be investigated and examined using cell biological and immunological methods. The characterization and optimization of delivery will be done in in vitro, using primary cultures of antigen-presenting cells, and in vivo using mouse models. The working delivery carrier will be then tested using viral nucleoproteins for anti-viral vaccines. The optimal vaccine delivery vehicle resulting from this project will be a non-viral/ non-bacterial delivery vector resembling a bacterial particle capable of invading cells and carrying immuno-stimulating/modulation signals recognized by immune surveillance mechanism(s), which is ideal for inducing robust, Th1-type, cell-mediated immunity. Targeted delivery carrier for robust vaccine formulations developed in this project will have great significances in human public health and nation's biodefense.